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Dec 1, 2016 - sient increase in ICP during exposure to a blast wave (138 kPa) generated in shock tube had a higher magnitude than the blast wave itself [19].
RESEARCH ARTICLE

Effects of Exposure to Blast Overpressure on Intracranial Pressure and Blood-Brain Barrier Permeability in a Rat Model Usmah Kawoos1*, Ming Gu1, Jason Lankasky1, Richard M. McCarron1,2, Mikulas Chavko1 1 Department of Neurotrauma, Naval Medical Research Center, Silver Spring, MD, United States of America, 2 Department of Surgery, Uniformed Services University of the Health Sciences and the Walter Reed National Military Medical Center, Bethesda, MD, United States of America * [email protected]

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Abstract

OPEN ACCESS Citation: Kawoos U, Gu M, Lankasky J, McCarron RM, Chavko M (2016) Effects of Exposure to Blast Overpressure on Intracranial Pressure and BloodBrain Barrier Permeability in a Rat Model. PLoS ONE 11(12): e0167510. doi:10.1371/journal. pone.0167510 Editor: Ma´ria A. Deli, Hungarian Academy of Sciences, HUNGARY

Exposure to blast overpressure (BOP) activates a cascade of pathological processes including changes in intracranial pressure (ICP) and blood-brain barrier (BBB) permeability resulting in traumatic brain injury (TBI). In this study the effect of single and multiple exposures at two intensities of BOP on changes in ICP and BBB permeability in Sprague-Dawley rats was evaluated. Animals were exposed to a single or three repetitive (separated by 0.5 h) BOPs at 72 kPa or 110 kPa. ICP was monitored continuously via telemetry for 6 days after exposure to BOP. The alteration in the permeability of BBB was determined by extravasation of Evans Blue (EB) into brain parenchyma. A significant increase in ICP was observed in all groups except the single 72 kPa BOP group. At the same time a marked increase in BBB permeability was also seen in various parts of the brain. The extent of ICP increase as well as BBB permeability change was dependent on intensity and frequency of blast.

Received: May 12, 2016 Accepted: November 15, 2016 Published: December 1, 2016 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are either within the paper or provided as supplemental information. Funding: This work was supported by Office of Naval Research (ONR) Work Unit 601152N.0000.000.A1308. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Introduction Blast induced traumatic brain injury (bTBI) has received much attention in the past few decades due to an increasing number of military personnel suffering from varying extents of brain injury [1]. TBI is the most prevalent type of injury in personnel involved in combat activity and most of TBI (63%) is caused by explosions[2]. Many war veterans exposed to blast exhibit symptoms of acute and chronic neurological deficits with serious impact on the quality of life and health care. There is insufficient information on the intensity of exposure which may cause mild or moderate TBI [3]. Much effort has been made to understand the biomechanics of TBI caused by blasts in laboratory environment by generating blast pressure waves in shock tubes [4–11]. This has provided a useful model to simulate blast effects and its consequences on physiology, neuropathology, and neurobehavior of animals for investigation in different laboratories [4]. The blood-brain barrier (BBB) is a heterogeneous selective permeability barrier composed of brain endothelial cells and tight junctions, which are pivotal in ensuring the integrity and selectivity of the barrier [12]. The immediate or primary effect of blast induced TBI involves the disruption in cerebral microvasculature and neighboring neuronal cells causing diffuse

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axonal injury, BBB breakdown, and brain contusions [13]. The delayed or secondary effects (such as inflammation) are initiated at a later point in time as a consequence of the primary damage [13]. The breakdown of BBB has been reported as a characteristic outcome after exposure to blast [12–17]. Skotak el al measured the extent of the BBB breakdown in rats at different levels of blast intensities after exposure to a single blast [18]. They found that the degree of BBB compromise 24 h following exposure to blast closely correlated with the intensity of BOP. In animals exposed to BOP IgG positive cells were seen in brain parenchyma, predominantly in cerebral cortex and hippocampus indicating leakage of markers through the BBB. The primary damage after TBI leads to a cascade of events causing cellular stress, inflammatory response, edema, and changes in intracranial pressure (ICP) [13]. TBI caused by BOP resulted in a transient sharp rise followed by a gradual increase in ICP [9, 18–21]. Interestingly the transient increase in ICP during exposure to a blast wave (138 kPa) generated in shock tube had a higher magnitude than the blast wave itself [19]. Time-course of ICP changes in groups of rats exposed to low-level blasts were reported by Saljo et al [22]. Under low levels of single blast exposure, ICP showed a slow-rising, sustained increase to a maximum level, following which it gradually declined to the normal levels [22]. Saljo et al reported a dependence of increase in ICP (peak and delay in elevation) on the intensity of blast when rats were exposed to a single blast of 10, 30, and 60 kPa [21]. Elevated ICP can be an early response after TBI and in cases where edema and contusions are seen ICP may continue to rise gradually [23]. Clinical experiences suggest that peak ICP elevation after impact TBI may occur 3–5 days after the insult [24]. In a prospective study of 201 TBI patients, intracranial hypertension was documented in 155 patients who had continuous high levels of ICP (> 20 mmHg) lasting at least 5 min [23]. The highest mean ICP was reached within 2 days in one-third of the cases; 3–4 days in another third. By day 5 (after injury) 80% of the cases had reached the maximum mean ICP. The effects of blast on BBB and ICP also appear to be dose-dependent. It was shown that low intensity blast induces early activation of oxidative and nitrosative reactions, which lead to BBB damage, consequent cerebral inflammation, and increase in ICP [15]. At higher blast intensity levels, BBB disruption seems to occur almost immediately followed by an increase in oxidative stress and the onset of neuroinflammation [8, 25]. Higher intensity blasts result in greater disruption in BBB than low intensity blasts. However the disruption caused by repeated blasts of same intensity may not be additive in nature [26]. In an in vitro BBB model there were no significant differences in the transendothelial electrical resistance (TEER) across a brain endothelial monolayer when comparison was drawn between single and double blasts of the same intensity [26]. Repeated blasting did not significantly reduce TEER, but the second exposure delayed TEER recovery in BBB cultures. In the present study, TBI caused by blast overpressure (BOP, generated in shock tube) was assessed by monitoring the changes in ICP and BBB permeability in a rat model. The response to single and repetitive blasts at two intensities was characterized.

Materials and Methods Animals and groups The study protocol was reviewed and approved by the Walter Reed Army Institute of Research/Naval Medical Research Center (NMRC) Institutional Animal Care and Use Committee in compliance with all applicable Federal regulations governing the protection of animals in research. Male Sprague-Dawley rats (weight = 300–350 g and age = 10 weeks at the time of experiment) were obtained from Taconic Farms, NY and were given at least one week to acclimatize in NMRC vivarium. The study was divided into two separate parts to evaluate the effect of blast(s) on ICP and BBB permeability. For each part, the rats were divided into

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four groups (n = 6 for ICP; n = 6 for BBB) and exposed to either single or repetitive BOP with a peak amplitude (±SD) of 72 ± 3 or 110 ± 3 kPa. The duration of the positive phase (overpressure) and impulse (integral over time) during the positive phase were 5.1 ms and 0.15 kPa.s for 70 kPa; and 7.1 ms and 0.32 kPa.s for 110 kPa blasts. Animals in the repetitive exposure groups were exposed to three consecutive BOPs separated by 0.5 h. A sham group not exposed to blast and treated the same way as the blasted groups (n = 5) was included for BBB permeability study. For ease of presentation, the groups are denoted as: 1x72 kPa, 1x110 kPa, 3x72 kPa, and 3x110 kPa.

Exposure to BOP Blast wave was generated in a compressed air-driven shock tube as described before [9, 27]. The static pressure wave characteristics were measured by a piezoelectric sensor (PCB Piezotronics, Buffalo, NY) placed next to the animal’s head. Animals were anesthetized with 5% isoflurane for 3 minutes and secured in a retainer inside the tube in frontal position relative to pressure wave propagation, approximately 1 foot from the open end of the tube.

ICP monitoring ICP was measured by Millar telemetry system [28] (Millar Instruments. Inc., Houston, TX) in freely moving animals, except for 3 min at the time of BOP exposure. The system consists of an ICP telemeter for pressure measurement and wireless transmission; SmartPad as a power supply and a wireless link to the telemeter; and PowerLab and LabChart (ADInstruments, Colorado Springs, CO) for data acquisition from SmartPad, recording and display. ICP data was collected at a sampling rate of 1k/s and a triangular window (n = 33) was used for smoothing. The telemeter is completely implantable and consists of a sensor-tipped catheter emanating from a body holding the electronics. The telemeter is designed in such a way that the sensor is placed in a target location and the body is implanted in the abdominal sac of the animal. The positioning of the telemeter body ensures adequate battery charging and seamless data transmission when the animal is placed on top of the SmartPad. For continuous monitoring of ICP, animals underwent a sterile surgical procedure for the implantation of ICP telemeter. ICP telemeter implantation. Animals were anesthetized with a mixture of Ketamine/ Xylazine (i.p., 70/4 mg/kg). A 2 cm incision was made in the lower right quadrant of the abdomen and the telemeter body was inserted behind the viscera and sutured to the internal abdominal wall. The sensor-tipped catheter was tunneled through the subcutaneous space over animal’s back and neck to later emerge from the incision made on its head. The abdominal incision was sutured and the animal was turned to the ‘sphinx’ position. The animal’s head was immobilized in a stereotaxic frame and a 1 cm incision was made on the dorsal midline of the scalp. The skin was removed to expose the bregma. A 1 mm hole was drilled in the bone using a tapered dental burr at 0.9 mm lateral from midline and 1.5 mm posterior to bregma. A 25-gauge needle was used to puncture dura for insertion of the pressure sensor-tipped catheter. The sensor was inserted to reach a depth of 3.5 mm below the surface of the skull in order to be positioned in the right lateral ventricle. The catheter was glued to the surface of the skull by Vetbond ™ (cyanoacrylate, Hanna Pharmaceuticals Supply Co. Inc., Wilmington, DE) and the scalp incision was sutured. After surgery animals received Ketorolac (5 mg/kg, subcutaneously), and they were allowed to recover from anesthesia. The proper placement of sensor and the response of telemeter were confirmed by observing an increase in ICP after compressing internal jugular vein and/or suspending the animal by its tail with the head in ‘dependent’ position. A period of 24 h was allowed for the stabilization of the telemeter before animals were exposed to blast. Animals were randomly assigned to groups for exposure to blast and

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ICP was continuously monitored for 6 days after the blast. At the end of study animals were euthanized with Euthasol (Virbac AH, Inc., Fort Worth, TX). Data analysis. For simplification of data analysis, representative data spanning over a time window of 3 h (Day -1, pre-blast; Day 1—Day 5, post-blast) or 4 h (Day 0, day of blast) were selected, with each window starting at the same time of day. The data are expressed as means ± SE and two-way ANOVA followed by Bonferroni post-hoc tests (with p